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Chapter 12. DNA Technology and Genomics. DNA and Crime Scene Investigations DNA fingerprinting has provided a powerful tool for crime scene investigators DNA is isolated from biological fluids left at a crime scene

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chapter 12

Chapter 12

DNA Technology and Genomics

slide2
DNA and Crime Scene Investigations
    • DNA fingerprinting has provided a powerful tool for crime scene investigators
      • DNA is isolated from biological fluids left at a crime scene
      • The technique determines with near certainty whether two samples of DNA are from the same individual
    • DNA technology—methods for studying and manipulating genetic material—plays significant roles in many areas of society
bacterial plasmids and gene cloning
BACTERIAL PLASMIDS AND GENE CLONING
  • 12.1 Plasmids are used to customize bacteria: An overview
    • Recombinant DNA technology: techniques for combining genes from different sources
    • Gene cloning: production of multiple identical copies of gene-carrying DNA
    • Genetic engineering: direct manipulation of genes for practical purposes
    • Biotechnology: use of organisms or their components to make useful products
slide5
Recombinant DNA technology uses plasmids, small, circular DNA molecules that replicate separately from a bacterial chromosome
    • Desired genes inserted into plasmids to form recombinant DNA
    • Plasmids inserted into bacteria
    • Foreign genes copied when recombinant bacteria multiply into a clone
le 12 01 3

LE 12-01-3

Bacterium

Cell containing gene

of interest

Plasmid

isolated

DNA

isolated

Gene inserted

into plasmid

Bacterial

chromosome

Plasmid

Recombinant DNA

(plasmid)

DNA

Gene of

interest

Plasmid put into

bacterial cell

Recombinant

bacterium

Cell multiplies with

gene of interest

Copies of protein

Copies of gene

Clone of cells

Gene for pest

resistance

inserted into

plants

Protein used to

make snow

form at higher

temperature

Gene used to alter bacteria

for cleaning up toxic waste

Protein used to dissolve blood

clots in heart attack therapy

slide7
12.2 Enzymes are used to "cut and paste" DNA
    • DNA from two sources cut by restriction enzyme at specific restriction sites
    • Resulting restriction fragments contain a double-stranded sequence of DNA with single-stranded "sticky ends"
    • Fragments pair at their sticky ends by hydrogen bonding
    • DNA ligase pastes the strand into a recombinant DNA molecule
le 12 02

LE 12-02

Restriction enzyme

recognition sequence

G

A

A

T

T

C

DNA

T

A

G

T

A

C

Restriction enzyme

cuts the DNA into

fragments

A

A

T

G

T

C

A

A

T

T

G

C

Sticky end

A

A

T

T

C

Addition of a DNA

fragment from

another source

G

G

C

T

T

A

A

Two (or more)

fragments stick

together by

base-pairing

C

G

G

A

A

T

T

A

A

T

T

C

C

A

A

G

A

T

T

C

T

T

A

G

DNA ligase

pastes the strand

Recombinant DNA molecule

slide10
12.3 Genes can be cloned in recombinant plasmids: a closer look
    • Bacteria take up recombinant plasmids from their surroundings and reproduce, thereby cloning the plasmids and the genes they carry
      • Isolate DNA from two sources
      • Cut both DNAs with the same restriction enzyme
      • Mix the DNAs, which join by basepairing
slide11
Add DNA ligase to bond the DNA
  • Put plasmid into bacterium by transformation
  • Clone the bacterium

Animation: Cloning a Gene

le 12 03

LE 12-03

Human cell

E. coli

Isolate DNA

from two sources

Cut both DNAs

with the same

restriction enzyme

Plasmid

DNA

Gene V

Sticky ends

Mix the DNAs;

they join by

base-pairing

Add DNA ligase

to bond the DNA covalently

Recombinant DNA

plasmid

Gene V

Put plasmid into bacterium

by transformation

Recombinant

bacterium

Clone the bacterium

Bacterial clone carrying many

copies of the human gene

slide13
12.4 Cloned genes can be stored in genomic libraries
    • Genomic library
      • Set of cloned DNA fragments containing all of an organism's genes
      • Fragments can be constructed and stored in cloned bacterial plasmids (plasmid library) or phages (phage library)
le 12 04

LE 12-04

Genome cut up with

restriction enzyme

Recombinant

plasmid

Recombinant

phage DNA

or

Bacterial

clone

Phage

clone

Plasmid library

Phage library

slide15
12.5 Reverse transcriptase helps make genes for cloning
    • Complementary DNA (cDNA), which contains only the genes that are transcribed by a particular type of cell, can be created using reverse transcriptase
      • Cell transcribes genes
      • RNA splicing removes introns
      • Single-strand DNA created from RNA with reverse transcriptase
      • Enzymes added to break down RNA
      • Second DNA strand synthesized
le 12 05

LE 12-05

Cell nucleus

Exon

Intron

Exon

Intron

Exon

DNA of

eukaryotic

gene

Transcription

RNA

transcript

RNA splicing

(removes introns)

mRNA

Isolation of mRNA

from cell and addition

of reverse transcriptase;

synthesis of DNA strand

Test tube

Reverse transcriptase

cDNA strand

Breakdown of RNA

Synthesis of second

DNA strand

cDNA of gene

(no introns)

connection
CONNECTION
  • 12.6 Recombinant cells and organisms can mass-produce gene products
    • Recombinant cells and organisms constructed by DNA technology are used to manufacture many useful products, chiefly proteins
      • Bacteria are usually the best vectors
      • Some eukaryotic cells are used
        • Saccharomyces cerevisiae fungus for brewing and baking
        • Mammalian cells for pharmaceuticals
connection20
CONNECTION
  • 12.7 DNA technology is changing the pharmaceutical industry
    • DNA technology is widely used to produce medicines and to diagnose diseases
      • Therapeutic hormones
        • Example: humulin, human insulin produced by bacteria
      • Diagnosis and treatment of disease
        • Example: analysis to identify HIV
      • Development of vaccines
restriction fragment analysis and dna fingerprinting
RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING
  • 12.8 Nucleic acid probes identify clones carrying specific genes
    • Detecting genes depends on base pairing between the gene and a complementary sequence on another nucleic acid molecule
    • A nucleic acid probe
      • Is a short, single-stranded molecule of radioactively or fluorescently labeled DNA or RNA
      • Can base pair with a desired gene in a library, thus tagging it

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

le 12 08

LE 12-08

Radioactive

probe (DNA)

A

C

C

G

T

A

Mix with single-

stranded DNA from

various bacterial

(or phage) clones

G

Single-stranded

DNA

C

A

T

G

A

T

T

C

A

T

C

C

C

A

G

A

T

G

T

C

A

C

T

G

G

A

T

A

G

T

A

T

T

C

Base pairing

indicates the

gene of interest

G

C

G

G

A

A

connection26
CONNECTION
  • 12.9 DNA microarrays test for the expression of many genes at once
    • DNA microarray assays can reveal patterns of gene expression in different kinds of cells
      • Isolate mRNA
      • Make cDNA from mRNA using reverse transcriptase
      • Apply cDNA (single-stranded) to wells
      • cDNA binds to corresponding gene; unbound cDNA is rinsed away; remaining DNA produces a glow
le 12 09

LE 12-09

DNA microarray

Each well contains DNA

from a particular gene

Actual size

(6,400 genes)

mRNA

isolated

Unbound

cDNA rinsed

away

Reverse transcriptase

and fluorescent DNA

nucelotides

Nonfluorescent

spot

Fluorescent

spot

cDNA applied

to wells

cDNA made

from mRNA

cDNA

DNA of an

expressed gene

DNA of an

unexpressed gene

slide28
12.10 Gel electrophoresis sorts DNA molecules by size
    • Gel electrophoresis uses a gel as a molecular sieve to separate nucleic acids by size or electrical charge
      • Longer macromolecules move through the gel more slowly than shorter macromolecules, resulting in a pattern of bands on the gel
le 12 10

LE 12-10

Mixture of DNA

molecules of

different sizes

Longer

molecules

Power

source

Gel

Shorter

molecules

Completed gel

slide30
12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences
    • Differences in DNA sequences on homologous chromosomes produce sets of restriction fragments that differ between individuals
      • Are called restriction fragment length polymorphisms (RFLPs)
      • Are of different lengths and will migrate different distances in an electrophoretic gel
      • Can be used as genetic markers
le 12 11a

LE 12-11a

Crime scene

Suspect

w

G

C

T

A

Cut

G

C

C

G

C

G

C

G

G

C

C

G

z

x

G

G

C

C

Cut

Cut

G

G

C

C

C

C

G

G

y

y

C

C

G

G

DNA from chromosomes

le 12 11b

LE 12-11b

1

2

Longer

fragments

z

x

w

Shorter

fragments

y

y

slide33
Restriction fragments can be used as DNA probes to detect harmful alleles
    • Patterns of normal and harmful alleles identified
    • Banding patterns compared with probe
le 12 11c 3

LE 12-11c-3

Restriction fragment preparation

I



I

Restriction

fragments

Gel electrophoresis



I

I

Blotting

Filter paper

Radioactive probe

Radioactive, single-

stranded DNA (probe)

Probe

I

Detection of radioactivity

(autoradiography)



I

Film

I



I

connection35
CONNECTION
  • 12.12 DNA technology is used in courts of law
    • Forensic science is the scientific analysis of evidence for criminal and other legal investigations
    • DNA fingerprinting requires only about 1,000 cells
      • Radioactive probes mark electrophoresis bands that contain certain markers
      • Produces a specific pattern of bands to compare to those of accused person
      • Highly reliable because odds of two people having identical DNA fingerprints are extremely small
le 12 12a

LE 12-12a

Blood from

defendant’s clothes

Victim’s

blood

Defendant’s

blood

connection38
CONNECTION
  • 12.13 Gene therapy may someday help treat a variety of diseases
    • Gene therapy is the alteration of an afflicted individual's genes
      • Where a disorder is due to a single gene, it is sometimes possible to replace the defective gene with a normal allele
      • To be permanent, the normal allele must be transferred to cells that multiply throughout a person's life, such as bone marrow cells
le 12 13

LE 12-13

Cloned gene

(normal allele)

Insert normal gene

into virus

Viral nucleic

acid

Retrovirus

Infect bone marrow

cell with virus

Viral DNA inserts

into chromosome

Bone marrow

cell from patient

Bone

marrow

Inject cells

into patient

slide40
Gene therapy
    • May one day be used to treat both genetic diseases and nongenetic disorders, but progress is slow
    • Raises both technical and ethical issues
slide41
12.14 The PCR method is used to amplify DNA sequences
    • The polymerase chain reaction (PCR) can be used to quickly clone a very large number of DNA copies for analysis
      • DNA sample mixed with DNA polymerase, nucleotide monomers, other ingredients
      • Mixture exposed to cycles of heating to separate the DNA strands
      • During each cycle, DNA replicates, doubling the amount
le 12 14

LE 12-14

Initial

DNA

segment

1

4

8

2

Number of DNA molecules

genomics connection
GENOMICS CONNECTION
  • 12.15 The Human Genome Project is an ambitious application of DNA technology
    • The Human Genome Project was begun in 1990 and is now largely completed
      • Initially involved three stages: genetic (linkage) and physical mapping of chromosomes, followed by DNA sequencing
      • Superseded by "shotgun" approach, going directly to stage 3
    • The data are providing insight into development, evolution, and many diseases
slide45
12.16 Most of the human genome does not consist of genes
    • Surprisingly, the haploid human genome contains only about 25,000 genes
    • About 97% of the human genome consists of noncoding DNA
      • Gene-control sequences
      • Introns
      • Noncoding DNA located between genes
slide46
Repetitive DNA-nucleotide sequences present in many copies
    • Teleomeres found at chromosome ends
    • Transposons ("jumping genes") that can move about within the genome
connection47
CONNECTION
  • 12.17 The science of genomics compares whole genomes
    • Genomics is the study of whole sets of genes and their interactions
      • As of 2005, the genomes of about 150 species had been sequenced
      • Besides being interesting in themselves, nonhuman genomes provide understanding of the human genome
    • Proteomics is the study of the full protein sets encoded by genomes
genetically modified organisms connection
GENETICALLY MODIFIED ORGANISMS CONNECTION
  • 12.18 Genetically modified organisms are transforming agriculture
    • Recombinant DNA technology can produce new varieties of plants and animals for use in agriculture
      • Genetically modified (GM) organisms have acquired genes by artificial means
      • Transgenic organisms have had genes from other organisms inserted into their genomes
    • A number of important crop plants are genetically modified using the Ti plasmid

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

le 12 18a

LE 12-18a

Agrobacterium tumefaciens

Plant cell

DNA containing

gene for desired trait

Ti

plasmid

Recombinant

Ti plasmid

Insertion of gene

into plasmid using

restriction enzyme

and DNA ligase

Introduction

into plant

cells in

culture

Regeneration

of plant

T DNA

T DNA carrying new

gene within plant chromosome

Plant with new trait

Restriction site

connection53
CONNECTION
  • 12.19 Could GM organisms harm human health or the environment?
    • Scientists have developed safety guidelines to minimize the risks involved in genetic engineering
      • Laboratory safety procedures
      • Organisms altered so they cannot live outside the lab
    • Exported GM organisms must be identified
    • Today most concern focuses on transgenic crop plants passing their genes to wild relatives
connection56
CONNECTION
  • 12.20 Genomics researcher Eric Lander discusses the Human Genome Project
    • Dr. Eric Lander founded the Broad Institute of MIT and Harvard
      • Uses genomics to develop new methods to investigate and treat diseases
    • The Human Genome Project
      • Results will give researchers the opportunity to examine the human genome from a "big picture" approach
      • Revolutionizing evolutionary biology